The ovarian hormones estradiol (E) and progesterone (P) have important effects on higher brain function besides their role in reproduction, and they affect many brain regions besides the hypothalamus, including the hippocampus and cerebral cortex. This project addresses molecular mechanisms by which E and P regulate spine synapse formation and maturation (SSF/M) in the hippocampus, a brain region involved in learning and memory of events in daily life. Synapse turnover occurs cyclically during the rat ovarian cycle by a mechanism that requires the activity of NMDA receptors and the participation of genomic and non-genomic estrogen (ER)and progestin receptors (PR).E-dependent SSF/M occurs in hippocampus and in prefrontal cortex in rhesus monkeys, as well as in rats and mice. There is electrophysiological, neuroanatomical and behavioral detail for this phenomenon, which has established its potential relevance for effects of E deprivation on human cognitive function. The two forms of the E receptor, ERa and ERp, as well as PR are found in both nuclear and non-nuclear sites in hippocampal neurons, and emerging evidence indicate that E and P affect non-genomic signalling pathways. Our central hypothesis is that E and P effects upon SSF/M and removal involve rapid non-genomic hormone actions, as well as direct and indirect genomic effects, leading to the modification of the actin cytoskeleton, as well as the stimulation of protein synthesis and coordinated genomic activity that leads to SSF/M. We shall address the role of ER subtypes in SSF/M and memory using specific agonists for each receptor isoform. Because mice are useful for genetic manipulations, we shall study key aspects of E and P action on SSF/M in mice. Studies will be carried out on mice lacking ERa and ER(3as well as on mice with an inhibitory form of the transcription regulator, CREB, that may normally mediate indirect genomic actions of E. We shall determine the role of E dependent signaling pathways in SSF/M based upon data we have obtained from in vitro studies of E-responsive NG108-15 cells in order to study hippocampal neurons in vitro using confocal microscopy and then in vivo using light and electron microscopy. We shall study non-nuclear signalling actions of E, namely, the regulation of phosphorylation of Akt and the phosphorylation of LIMK-1 leading to cofilin phosphorylation and actin polymerization. Finally, since E-regulated spine SSF/M is reversed rapidly by P, we shall investigate how P causes loss of spines via the ubiquitin-proteasome pathway. These studies will provide new basic information to better understand and treat cognitive and other higher brain functions that are affected by surgical- or age-related loss of ovarian hormones during menopause, as well as protection of the brain from seizures and stroke by E and P.